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Thromb Haemost 2025; 125(10): 985-997
DOI: 10.1055/a-2474-5644
Cellular Haemostasis and Platelets

Roles of ROCK/Myosin Pathway in Macrothrombocytopenia in Bernard–Soulier Syndrome

Authors

  • Ponthip Mekchay

    1   Division of Hematology, Faculty of Medicine, Excellence Center in Translational Hematology, Chulalongkorn University, Bangkok, Thailand

  • Praewphan Ingrungruanglert

    2   Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

  • Netchanok Leela-adisorn

    2   Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

  • Noppacharn Uaprasert

    1   Division of Hematology, Faculty of Medicine, Excellence Center in Translational Hematology, Chulalongkorn University, Bangkok, Thailand

  • Nipan Israsena

    2   Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

  • Ponlapat Rojnuckarin

    1   Division of Hematology, Faculty of Medicine, Excellence Center in Translational Hematology, Chulalongkorn University, Bangkok, Thailand

Funding This research was supported by the Thailand Science Research and Innovation Fund (Fundamental Fund), Chulalongkorn University. P.M. was supported by Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University.
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Abstract

Background

Megakaryocytes (MK) from Bernard–Soulier syndrome (BSS) induced pluripotent stem cells (iPSCs) yielded reduced numbers but increased sizes of platelets. The molecular mechanisms remain unclear. This study aims to determine roles of signaling molecules involved in this process.

Material and Methods

Wild-type (WT) iPSCs and iPSCs from BSS patients with GP1BA (BSS-A) or GP1BB (BSS-B) mutations were differentiated into MKs and platelets with or without myosin II inhibitor (blebbistatin), ROCK inhibitor (Y27632), and procaspase-3 activator (PAC-1). Proplatelet and platelet numbers and sizes were characterized. The iPSC lines containing tubulin-green fluorescent protein (GFP) reporters were constructed to observe proplatelet formation under time-lapse microscopy.

Result

BSS-derived MKs (BSS-MKs) yielded fewer but larger platelets compared with the WT. In the presence of blebbistatin, ROCK inhibitor, or PAC-1, WT, BSS-A, and BSS-B MKs could generate more platelets with decreased sizes, but PAC-1 caused CD42 loss on WT platelets. The proportions of proplatelet formation from MKs carrying tubulin-GFP were not different between WT and BSS-MKs, as well as among inhibitors. Notably, initially thick cytoplasmic processes were transformed into thin branching proplatelets over the observation time. The proplatelet shafts of BSS-MK became thinner in the presence of blebbistatin or ROCK inhibitor, but not of PAC-1, which displayed uneven F-actin distribution.

Conclusion

Inhibition of the ROCK/myosin pathway, downstream of GpIb, could restore normal morphology of proplatelets in BSS-MKs. Procaspase-3 activation could increase platelet yields, but with abnormal proplatelet and platelet structures. Our model can be used for therapeutic drug screening and a disease model for platelet production in the future.

Keywords

megakaryocytes - Bernard–Soulier syndrome - platelet formation - induced pluripotent stem cell

Authors' Contribution

P.M. designed the overall research, performed the experiments, analyzed data, interpreted and discussed the results, and wrote the manuscript; P.I. and N.L. performed the experiments and analyzed data; N.U. interpreted and discussed the results; N.I. designed the overall research, interpreted, and discussed the results; P.R. designed the overall research, analyzed data, interpreted and discussed the results, and wrote the manuscript. All the authors reviewed and approved the manuscript.




Publication History

Received: 10 September 2024

Accepted: 17 November 2024

Article published online:
18 December 2024

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